Screening of genetic variations has become a matter of increasing importance in all types of genetic analysis, including medical analysis. Screening techniques have become common for detecting gross genetic variations, such as restriction fragment length polymorphisms. However, genetic variation can also be cause by subtler changes in the genetic code, including changes in a single nucleotide in a particular sequence, referred to as single nucleotide polymorphisms (SNPs).
Among the areas where such genetic variations can be of critical importance is the treatment of Acquired Immunodeficiency Syndrome (AIDS). AIDS is caused by Human Immunodeficiency Virus type-1 (HIV-1) which can be subdivided into three highly divergent groups that include: M (main), O (outlier), and N (non-M or O). HIV-1 group M strains are responsible for over 95% of infections worldwide and are further separated into at least nine discreet subtypes or clades (A, B, C, D, F, G, H, J, and K), based on the sequence of complete genomes. Additionally, 13 recombinant forms (CRF) have been characterized that further increase the growing HIV-1 diversity. Overall HIV-1 displays 15-40% nucleotide diversity between subtypes and up to 30% nucleotide diversity within a subtype. Additionally, it has been estimated that there can be between 5 and 10% sequence diversity within an infected individual. In the past few years, HIV-1 research on pathogenesis, replication and host-virus interaction has shifted focus from subtype B laboratory strains to primary HIV-1 isolates of all subtypes. Thus, the heterogeneity of HIV-1 has introduced new challenges for cloning and subsequent functional studies.
HIV-1 carries a genome consisting of ribonucleic acid (RNA) rather than deoxyribonucleic acid (DNA). In addition to the same core gene structure shared among all retroviruses (i.e. the gag, pol, and env genes), the HIV-1 genome also harbors several genes found in multiply and singly spliced RNA transcripts (i.e. vif, vpr, tat, rev, vpu, and nef) that encode for several accessory proteins. Standard molecular biological techniques for manipulation of HIV-1 genetic elements are difficult to apply due to poor sequence conservation between different isolates. Unique restriction endonuclease sites are not conveniently distributed across the HIV-1 genome for selective introduction or mutation of various regions or genes. Additionally, the insertion of new restriction sites for cloning is problematic due to the likely disruption of one or more of the multiple open reading frames found in the virus. As a result, current research on HIV-1 replication relies upon a few closely related molecular clones that have matching restriction endonuclease sites. Alternatively, other methods for studying HIV-1 genes involve trans gene expression with respective deletion in a molecular clone to create pseudotyped viruses. However, these pseudotyped viruses are limited to a single round of replication since the full length functional genome is not packaged in the virus particle.
Treatment of individuals infected with HIV-1 with antiretroviral drugs (ARVs) has changed the face of the AIDS epidemic. Previously, all infection with HIV-1 led to AIDS and mortality in an average of two to seven years. The first anti-HIV-1 ARV, 3′-azido-3′-deoxythymidine (AZT, zidovudine, Retrovir®) was approved in 1987 for therapy but was largely unsuccessful in prolonged treatment due to resistance. Until the advent of triple drug combination therapy (Highly Active AntiRetroviral Therapy or HAART), drug resistance was common in all treated patients and remained the primary reason for the failure of ARVs to control HIV viremia. Due to the issues of adherence, the need for lifelong therapy, drug tolerance, and incomplete viral suppression, resistance to ARV still emerges in patients undergoing HAART. Unfortunately, ARV resistance triggers a resumption of disease progression unless new ARVs can be administered in a HAART regimen. Pharmaceutical companies have been successful in continually developing new ARV and in different drug classes.
There are now FDA-approved drugs sub grouped into three classes of anti-HIV ARVs, which target different steps in the HIV lifecycle: reverse transcriptase inhibitors (RTIs) (nonnucleoside (NNRTI), and nucleoside (NRTI)), protease inhibitors (PRIs), and entry inhibitors (EI) (enfutride, fuseon or T20). Several new HIV-1 entry inhibitors that occlude a viral receptor on the host cells have been effective in pre-clinical development and are now in advanced clinical trials. Additionally, Integrase, another catalytic enzyme of HIV-1 has also been recognized as a rational therapeutic target for the treatment of infection. Integration of the HIV-1 proviral DNA genome into the host genome is essential for viral mRNA transcription but also establishes a stable viral episome in the host genome. Integrase inhibitors and various derivatives could be on the cusp for phase III clinical trials and FDA approval for use in HAART regimens. The continual need for new HIV-1 inhibitors targeting new enzymes or viral processes is due to the emergence of primary resistance to the current PRI and RTIs licensed for therapy. Many of the drug resistant HIV-1 strains selected under a previous regimen also confer cross-resistant to other ARVs in the current FDA-approved arsenal. Cross-resistance limits the use of other drugs in salvage therapy (i.e. following resistance to the first line regimen). Thus, monitoring drug resistance has become a key clinical tool in the management of HIV infected patients by their physicians.
HIV infection of CD4+ cells is facilitated by and dependent upon viral envelope (env) glycoprotein gp120 interaction with cellular receptor CCR5 or CXCR4. After CD4 binding, the gp120 bridging sheet subdomain makes important contacts with the chemokine receptor N-terminal domain. A second subdomain in gp120, the variable subdomain 3 (V3), is the main determinant of coreceptor usage, making contacts with the second extracellular loop (ECL2) of either CCR5 or CXCR4. An HIV-1 virion may use CD4 and either CCR5 (in which case the virus is denoted as an R5-tropic virus) or CXCR4 (denoted an X4-tropic virus) for entry, but may have capability to use both. Most new patient infections involve R5-tropic variants, even when the dominant variant in the infecting partner has dual/mixed tropic variants, but X4-tropic variants emerge in over 50% of patients who progress to AIDS. Since X4 capability is associated with disease progression, it is thought to represent a more aggressive phase in the course of natural HIV-1 infection, corroborated by in-vivo models demonstrating the more pronounced cytopathic effects of X4-tropic strains. While the factors which influence coreceptor switching during the natural course of clinical HIV-1 infection are not well understood, tropism measurements remain a useful tool in management of HIV+ patients, providing valuable information about both clinical disease progression and treatment options.
Characterization of the HIV-1 entry process has led to development of entry inhibitors, including Maraviroc (MVC), a CCR5 antagonist, a small molecule which binds the CCR5 coreceptor in such a way that CCR5 interaction with gp120 is precluded. Maraviroc was approved for use in treating HIV in 2006. In the MVC-bound form, CCR5 ECL2 is unable to make the necessary contacts with gp120 V3 subdomain. In the two years since MVC approval, viral tropism has gained more attention than ever before because MVC use is contraindicated for individuals harboring X4-tropic viruses and, in fact, might select for more pathogenic X4-tropic variants. Maraviroc therapy decisions, particularly for patients failing a front-line medication, must then be guided in part by measurement of the X4-tropic component of the patient infection.
Amino acids 11 and 25 in the HIV-1 surface glycoprotein gp120 V3 subdomain are thought to be the primary determinants of HIV-1 coreceptor usage. If the identity of either amino acid 11 or 25 is one bearing a positive charge, it is highly probable that the isolate is capable of using CXCR4 for entry into cells. Therefore, there is a need for an assay method for determining the identity of amino acids 11 and/or 25 of the HIV-1 surface glycoprotein gp120 V3 subdomain.
Available assays for functional tropism assessment using tissue culture methods are available, but they are slow and expensive. Commercially available genetic tests are either insensitive or unreliable. Thus, new methods are needed for detecting a number of single nucleotide polymorphisms particularly those that have been recognized as conferring drug resistance.
Monogram Biosciences is one company which provides tropism measurement, marketed as the Trofile assay. Bacterial recombination is used to insert PCR-amplified patient gp160-encoding sequences into an envelope expression vector. The resulting construct is co-expressed with a packaging construct, a plasmid which contains a packaging signal and expresses the remaining HIV proteins and luciferase, in an eukaryotic cell line. Expression of both plasmids creates virus-like particles (VLPs) coated with patient-derived gp120/gp41 trimers. Supernatants from the co-expression cultures, enriched in VLPs, are used to infect target cells expressing heterologous human CD4 and CXCR4. VLPs with envelope glycoproteins derived from X4-tropic patient viruses can enter the target cells and integrate the luciferase ORF. X4-tropism is assessed quantitatively by luminometry after incubation of the target cell lysates with a pro-luminescent luciferase substrate. Other assays have also been reported using similar methods for generating recombinant virus constructs and coreceptor determination yielding results comparable to the Monogram test. Although this functional assay has been instrumental in determining which patients are not candidates for maraviroc therapy, it is expensive and the nature of the test dictates a present turnaround time of at least 14 days. As with functional HIV drug resistance assays, this has fueled a demand for faster and less expensive predictive testing.
Genotyping and prediction using bioinformatic algorithms may be more convenient and accessible than tropism phenotyping, but the methods for doing so have a common caveat in that all rely on DNA sequencing data. Treatment-experienced patients often have mixed populations of some X4 and some R5-tropic viruses which are missed by bulk sequencing. While bioinformatics approaches might be faster and less expensive than functional tropism assignment, the insensitivity of DNA sequencing for minor variants is a significant handicap to their predictive value. One technique circumvents the DNA sequencing problem intrinsic to bioinformatic algorithms by detecting differential electrophoretic migration of patient-derived sequences hybridized to a standard probe sequence in a native polyacrylamide gel. Patient V3 loop sequences are PCR amplified, denatured and annealed to a short radiolabeled PCR product from a standard X4-tropic variant. Heteroduplexes are resolved by electrophoresis and differ in electrophoretic mobility depending on their complementarity to the probe sequence. In one recent study, an X4-positive result by the commercial assay (SensiTrop) had high predictive value (most X4 calls were correct), but was unable to call X4 phenotypes for over 50% of samples shown to be dual/mixed or X4-tropic by a functional assay. It seems that sensitivity issues resolved by circumventing bulk DNA sequencing have been replaced by sensitivity issues of another sort for this application.
The use of an oligonucleotide ligation assay (OLA) to detect single nucleotide polymorphisms has been previously described. Typically, a fragment of nucleic acid of interest is amplified by polymerase chain reaction (PCR). The PCR product is then denatured and one polynucleotide (or primer) is selected from each of two sets of adjacent single-stranded polynucleotides, which are complementary to the PCR product. The first set of polynucleotides are essentially identical except for the last two nucleotides on the 3′ end of the polynucleotide. The second set of polynucleotide are essentially identical except for the first nucleotide on the 5′ end. In this way, a nucleotide sequence pertaining to a predicted and specific amino acid can be inferred through specific annealing and ligation of these two oligonucleotides over a codon. Each of the polynucleotides in the second set of polynucleotides are also typically labeled in some way, such as radioactive labeling or by covalent attachment of a molecule which will permit capture and identification of the presence of the polynucleotide, such as biotin. Once annealed to the PCR product, a ligase enzyme is used to join adjacent single-stranded polynucleotides. The ligase will only join polynucleotides where there are no mismatches between the PCR product and the last two nucleotides of the first single-stranded polynucleotide and the first nucleotide of the second single-stranded polynucleotide. In other words, the polynucleotides will only be ligated where the combined single-stranded polynucleotides are completely complementary to the PCR product, particularly in the region of the junction of the two polynucleotides. Various combinations of the first and second polynucleotides are used in an OLA in a well of a streptavidin-coated 96-well plate. Thermostable ligase may be used and the ligation reaction may be followed by denaturation of the product, and further followed by additional ligation reactions. OLA product may then be analyzed and quantitated by colorimetric analysis of the plate well.
Suspension arrays are a new technology by which multiple analytes in a mixture can be measured independently. The Luminex flow fluorimeter (or suspension array analyzer) is functionally equivalent to a three-color flow cytometer with dedicated gating specifically designed for analysis of microscopic beads. It measures the bead-associated fluorescence intensity of two classifier fluorophores in the bead as well as a reporter fluorophore. In this new methodology described below, oligonucleotide ligation assay (OLA) ligation products may be quantified on Luminex beads if one oligonucleotide (the downstream or reporter capture oligonucleotide; RCO) is labeled with the reporter fluorophore and the other (the upstream or interrogator oligonucleotide) specifically associates with suspension array bead. A physical link (a template sequence) between two oligonucleotides then couples ligase recognition of the allele to bead enrichment with the reporter fluorophore. In an assay for minority single nucleotide polymorphisms (SNPs), however, the oligonucleotide reagents must be significantly consumed during ligase discrimination to elicit a detectable response on the suspension array. Additionally, assay sensitivity was previously insufficient for the detection of SNPs of low frequency in a population.
A need exists for suspension array-based single nucleotide polymorphism (SNP) screening system that may be used for a variety purposes, including the detection and quantitation of minority SNPs in a population, such as in HIV treatment.